Figure 3.

TAAR1 increases proliferation via cAMP-dependent Raf/MEK/ERK signaling in Ins-1 cells. A, Western blots of ERK1/2 phosphorylation in response to T₁AM (10 μm) and forskolin (0.3 μm) for 5–30 min in the presence (11 mm) or absence of glucose. B–F, addition of 10 μm MDL-12,330A (B), 150 nm PKA Cat-α siRNA (C), 10 μm Esi-09 and 30 μm Esi-05 (D), 150 nm Epac siRNA (E), or 10 μm AZ-628 (Raf inhibitor) and 50 μm PD98059 (MEK1/2 inhibitor) (F) inhibits ERK1/2 phosphorylation (10 min) induced by T₁AM and forskolin. Representative blots of pERK1/2 from one of at least three independent experiments are shown; blots were stripped and reprobed with total ERK1/2 as a loading control. G, quantitative PCR of B-Raf and C-Raf gene expression in Ins-1 β-cells. Data are expressed as relative mean ΔΔCT (± S.D.) compared with B-Raf mRNA levels, using Gapdh as the housekeeping gene, and were analyzed by Student's t test (***, p < 0.001). H, T₁AM (10 μm) and forskolin (0.5 μm) increase [³H]thymidine incorporation into Ins-1 cells (24 h), which is blocked by PD-98059 (50 μm) and AZ-628 (10 μm). Data were analyzed using one-way ANOVA (global p values are shown), with Dunnett's multiple comparisons post hoc test to determine significance between relevant groups (n = 6). *, p < 0.05; **, p < 0.01; ***, p < 0.001.